HIRI is a significant clinical complication frequently encountered during liver transplantation, hepatic resection, and major liver trauma (de Oliveira and Gonçalves, 2025). This condition is characterized by initial ischemic insult followed by reperfusion-induced oxidative stress, extensive inflammatory responses, and hepatocyte apoptosis, which collectively lead to graft dysfunction, prolonged hospital stays, and potential organ failure (Vardanian et al., 2008). Despite considerable progress in understanding the underlying pathophysiology of HIRI, effective therapeutic interventions remain scarce, underscoring an urgent unmet clinical need for novel protective strategies (George et al., 2024).

Cistanche deserticola Y.C. Ma, a renowned traditional Chinese herbal medicine in the family Orobanchaceae, has long been used owing to its diverse pharmacological activities, including anti-fatigue, neuroprotective, anti-aging, and immunomodulatory effects (Liu et al., 2025). In accordance with the Chinese Pharmacopoeia, the dried succulent stems constitute the official medicinal part of C. deserticola, and this was also the source material for the extract used in our study. Cistanche deserticola is primarily distributed in the arid and semi-arid regions of northwestern China—including Xinjiang, Inner Mongolia, and Gansu—and is also found in parts of Mongolia and Central Asia. Its ecological adaptation to desert environments has contributed to its traditional use and long-term medicinal development. In traditional Chinese medicine, the dried succulent stems of C. deserticola—recorded as Cistanches Herba (Rou Cong Rong)—are prescribed to “tonify the kidneys, nourish the essence, and moisten the intestines,” and are clinically used for syndromes related to kidney-yang deficiency such as fatigue, impotence, infertility, and senile constipation (Fu et al., 2018; Li et al., 2016). Among commercial preparations, the botanical name Cistanche deserticola Y.C. Ma was verified using The Plant List (http://www.theplantlist.org).

Phytochemical studies have revealed that C. deserticola contains abundant phenylethanoid glycosides (PhGs), which represent its principal bioactive constituents. The predominant glycosides include echinacoside and acteoside (verbascoside), together with isoacteoside, cistanoside A–F, and 2′-acetylacteoside, all of which have been repeatedly isolated from the dried stems of the plant (Xiong et al., 1998; Lei et al., 2020; Gao et al., 2015; Li et al., 2016). These compounds account for the characteristic chemical profile of C. deserticola stem extracts and contribute to their antioxidant, neuroprotective, and hepatoprotective properties (Wang et al., 2022; Yan et al., 2023).

Modern pharmacological investigations have further confirmed that total glycosides of C. deserticola mitigate chemical- or alcohol-induced liver injury, supporting their potential against hepatic ischemia–reperfusion injury (Xiong et al., 1998; Wang et al., 2022; Yan et al., 2023). However, the precise molecular mechanisms through which GCs exert hepatoprotective effects remain to be elucidated.

In recent years, cAMP signaling pathway has emerged as a pivotal regulator in cellular responses to oxidative stress and inflammation, essential components of HIRI pathogenesis (Mayr and Montminy, 2001). Among its downstream effectors, EPAC2, a guanine nucleotide exchange factor, has drawn considerable interest due to its protein kinase A (PKA)-independent roles in modulating various physiological processes, such as cell adhesion, survival, metabolic regulation, and oxidative stress resistance (De Rooij et al., 2000; Kawasaki et al., 1998; Stokman et al., 2014). Nevertheless, the specific involvement and functional significance of EPAC2 within the context of HIRI and whether it participates in the hepatoprotective action of GCs remain largely unexplored.

Rap1, a small GTPase and classical downstream effector of EPAC2, has been reported to modulate numerous signaling pathways, including the critical Hippo signaling pathway (Chang et al., 2018). The Hippo pathway, composed of Mammalian sterile 20-like kinase 1/2 (MST1/2) and Large tumor suppressor kinase 1/2 (LATS1/2) kinases, serves as a fundamental regulator of cell proliferation, tissue regeneration, organ size, and homeostasis (Bao et al., 2011). The downstream transcriptional effector YAP, upon dephosphorylation, translocates into the nucleus, activating protective gene transcription programs involved in liver regeneration, oxidative defense mechanisms, and autophagy induction (Wang et al., 2020). Indeed, YAP-mediated transcription promotes the expression of essential autophagy-related proteins such as Autophagy-related 5 (ATG5), Autophagy-related 7 (ATG7), and Beclin1 (Sun et al., 2021).

Concurrently, the JNK pathway, belonging to the mitogen-activated protein kinase (MAPK) family, is critically implicated in hepatic stress responses during ischemia–reperfusion injury (Wei et al., 2008). Persistent JNK activation is typically associated with enhanced inflammation, tissue injury, and necrosis, whereas transient JNK activation supports beneficial cellular adaptations, including the promotion of autophagy and stress tolerance (Zhou et al., 2015). Mechanistically, JNK-mediated autophagy has been reported to occur through phosphorylation of B-cell lymphoma 2 (Bcl-2), releasing Beclin1 to facilitate autophagic flux, or through direct transcriptional upregulation of autophagy-related genes (Yang and Yao, 2015).

In this study, we explored the hypothesis that GCs could mitigate liver injury and apoptosis in HIRI by activating a novel cAMP–EPAC2–Rap1 signaling cascade that subsequently triggers the Hippo–YAP and JNK pathways. Utilizing both in vivo and in vitro models of HIRI, we aimed to clarify the protective molecular mechanisms underlying the effects of GCs. These findings not only enhance our understanding of the mechanistic basis of GCs but also identify the EPAC2–YAP–JNK signaling axis as a promising therapeutic target for future interventions in managing HIRI.